1. Field of the Invention
This invention is directed to articles of manufacture and to analytical methods. More specifically, this invention resides in the provision of a highly effective delivery system for dry chemistry reagents; and, in the incorporation of this delivery system into a plurality of diverse test strips. It is understood that the phrase "dry chemistry reagents" is inclusive of reagent systems for both clinical chemistry assays and immunoassays. This dry chemistry reagent system is useful in the rapid and efficient analysis of biological fluids. One of the unique features of this system is its ability to accomplish such analysis of samples containing cellular and particulate matter or macromolecules without prior seperation of such endogenous materials from the sample. Such separation is generally desirable, and can be essential in certain analysis to prevent such materials from masking detection of a reporter molecule which is indicative of the presence of the analyte of interest.
2. Description of the Prior Art
The search for a simple and effective means for performance of analytical testing of heterogenous fluid samples, notably biological samples, has spanned more than twentyfive years. Much of the early work in this area was directed to the development of a reagent format which would be compatible with a simple yet effective analytical protocol. Probably one of the simpler of these systems was based upon the provision of a "dry reagent chemistry system"; that is, a reagent system that was imbibed into an absorbent medium, dried and reconstituted by simple addition/absorption of the fluid sample by the absorbent medium.
The patent and technical literature is voluminous regarding the development and refinement of such dry chemistry reagent systems. These systems have traditionally included the reagent coated tube technology, the reagent coated bead technology and the so-called "paper" or "bibulous" layered systems. A format of dry chemistry reagent system which continues to enjoy increasing popularity is based upon single and multi-layered bibulous media.
One of the earlier and more successful of adaptations of the bibulous media format for dry chemistry reagent systems involved the development of a series of assays for analysis of whole blood. Some of the earlier patents in this field described the adaptation of this format to analysis of heterogenous fluids (whole blood) for glucose and other common analytes (i.e. urea, cholesterol) of interest. The following list is representative of the patent literature directed to simple analytical devices (test strips) illustrating the adaptations of dry chemistry reagent system to analysis of heterogeneous biological fluids: U.S. Pat. Nos. 3,061,523 (to Free); 3,552,925 (to Fetter); 3,607,093 (to Stone); 3,092,465 (to Adams); 3,298,789 (to Mast); and 3,630,957 (to Rey).
U.S. Pat. No. 3,061,523 (to Free), describes the basic chemistry reagent system which has become the standard for colorimetric determination of glucose in biological samples. The chemistry system described by Free is contemplated for use in conjunction with a "dip stick" test. In a typical configuration of the Free invention, a solid phase (i.e. sticks or test strips) is pre-treated with his novel chemistry formulation. The reagent treated portion of this solid phase can thereafter be contacted with a sample suspected of containing glucose. The intensity of color which is developed as a result of such contact is compared to a control or standard and a semi-quantitative determination of glucose level in the sample thereby computed.
In the specific embodiments of the Free device, the dry chemistry reagent system is prepared by first dissolving the reactive constituents in a gelatin base and thereafter impregnating strips of filter paper with this dry chemistry reagent system. This is achieved by simply immersing the filter paper in the gelatin base/reagent system for a sufficient interval to effect impregnation of the reagents into the filter paper. The filter paper treated in this fashion is thereafter dried. The gelatin component of the impregnating solution is reportedly essential to the uniformity of color development. Presumably, the presence of the gelatin controls or inhibits the migration of fluids within the filter paper, thereby minimizing chromatographic separation of reagents and/or sample.
A drop of blood (preferably whole blood) is then applied to the portion of the filter paper containing the dry chemistry reagent system; allowed to react with the reagents contained therein (for approximately 60 seconds) and, thereafter the blood (presumably the red cells) rinsed from the paper. The intensity of the color indicator which is developed as a result of the interaction of the glucose and the reagents within the paper, is thereafter observed or measured. The recommendation (if not a requirement), of the Free system, that the red cells be rinsed from the surface of the test strip, implies that their removal is desirable, if not essential, to observation/measurement of the colored indicator. The rinsing of the red cells from the test strip can be expected to introduce analytical error into such analysis, thus, impairing the accuracy of the test results.
Where the technician performing this test is dealing with a patient sample that may contain infectious microorganisms or viruses, the requirement that the sample be rinsed of red blood cells unnecessarily exposes the technician to potential infection.
U.S. Pat. No. 3,552,925 (to Fetter), represents an improvement to the glucose test element described in the Free patent. Fetter discloses a method and device for effectively separating the whole blood sample into its serum components and into its erythrocyte components (red blood cells and other color forming constituents). Fetter achieves this separation by treatment of a defined area of his sample collection device on his test element with certain water soluble salts. The contact of the whole blood sample with these salts in the test element results in the localized reaction of the erythrocytes (and the other colored components of the whole blood) with these salts and the resultant separation of the serum component therefrom. The serum fraction is, thus, free to migrate or diffuse into the test element. The migration and/or diffusion of the serum component is generally via capillary action or some other passive transport mechanism. The manner in which the sample is applied and the nature of the test medium, effectively transports and distributes the serum to another defined chemically reactive area of the test element containing test reagents. The test reagents of the test element are specific for one or more analytes of interest in the serum fraction (i.e. glucose, galactose, urea, uric acid, phenylalinine and/or various enzymes).
The various configurations of the Fetter test element contemplate a single laminae (FIGS. 1 and 8), having discrete areas of chemical treatments; or a multi-layered structure, wherein a single chemical treatment is confined to each of the layers of the laminate (FIGS. 3 through 7).
Fetter also indicates that the same matrix can be used to retain both the separating reagent and the reagent specific for the analyte of interest. In this latter embodiment of his invention, the whole blood sample would be applied to one side of the strip held in the horizontal position. After adequate penetration of the sample into the matrix containing both the separating and test reagents, the test strip would be inverted and color development observed (if any) on the site opposite the site of application of the whole blood sample.
Fetter is not apparently concerned with potential interference of the colored blood components with the development and/or observation of the indicator species. It is, however, apparent that at low concentrations of analyte, the highly colored blood components could interfere and/or mask the presence of the indicator from visual observation/detection.
Canadian Pat. No. 659,059 represents one of the earlier attempts at preparation of unitary dry chemistry reagent system in a single layer format. The dry chemistry reagent system of this patent is based upon the preparation of a common slurry containing the reactive components of the system along with the ingredients of the bibulous medium (i.e. cellulose acetate). The resultant slurry is then cast and dried. The reactive components of the system are physically entrapped within the resultant film/coating. The physical nature of the film/coating lends itself to absorption of a fluid sample without release (leaching) of the reactive components.
U.S. Pat. No. 3,092,465 (to Adams) describes a diagnostic device for detection of glucose in a heterogenous biological fluid sample (i.e. blood or urine). The device consists of a bibulous medium containing a dry chemistry reagent system specific for reaction with a glucose; and, the manifestation of such reaction by production of a visible change in color. This bibulous medium is provided with a "protective coating" in the form of a semi-permeable membrane. This membrane is selective for the exclusion of larger molecules (i.e. proteins and hemoglobins) while freely allowing the fluid fraction containing the glucose to be absorbed by the bibulous medium. This protective coating thus allows the test strip supporting the dry chemistry reagent system to be dipped or immersed in the test fluid without absorption of the colored components of the sample. The protective coating of the sample receptive surface can then be wiped free of an interfering particulate or colored matter prior to monitoring for color change. The wiping of the surface of the analytical element can introduce analytical error into such analysis, thus, impairing the accuracy of the test results. Similar error is to be expected if such debris were removed by washing or other physical means.
U.S. Pat. No. 3,298,789 (to Mast) describes a test article for detection of glucose in heterogenous fluid samples (i.e. whole blood). This test article is composed of a layer of bibulous material impregnated with a dry chemistry reagent system. This layer is overcoated with a smooth semi-permeable film of transparent ethyl cellulose. The test device is used to detect glucose in whole blood by simply applying a whole blood sample (2-3 drops) on the surface of the semi-permeable film coating which has been applied to the reagent impregnated material. After a brief interval, the fluid fraction of the sample is absorbed by the bibulous material. The cellular (colored) fraction of the sample is then wiped off the surface of the semi-permeable film to allow for observation/measurement of the indicator produced by reaction of the glucose and reagents within the bibulous layer. The surface characteristics of this semi-permeable film are thus critical to the operation of this device in the analytical environment. More specifically, the degree of smoothness of the protective film (surface porosity) is critical and must be sufficiently fine to avoid penetration of the cellular components and hemoglobin fractions into the surface of the film. As noted in the discussion of the Free and Adams patents, the removal of cells and colored debris from the sample receptive surface of the test element can introduce analytical error into such analysis.
U.S. Pat. No. 3,607,093 (to Stone) describes a liquid permeable membrane, of uniform chemical composition, having within its matrix, a dry chemistry reagent system. The membrane selected by Stone for his device is similar in its surface characteristics to the protective film of Mast. The analytical protocol utilizing the Stone device is also similar to that of Mast and requires the physical wiping of the sample receptive surface of the membrane for removal of cellular debris and colored materials (from the sample) to allow for observation/measurement of a reaction product indicative of the presence of the analytes of interest. The Stone analytical system suffers from the same deficiency which is common to Free, Mast and Adams, previously discussed; namely, the introduction of analytical error by required physical removal or washing of the cellular and colored debris from the sample receptive surface of the analytical element, prior to monitoring for indicator development.
Additional modification and enhancements have been made to the basic dry reagent chemistry formats described above. These modifications and enhancements have focused upon providing multiple test zones on a common test element; increasing the precision and correlation of indicator development with concentrations of analyte; and, greater control in absorption/distributions of the sample within the bibulous reagent impregnated medium. The following patents are illustrative of the enhancements and modification the dry reagent chemistry format: U.S. Pat. Nos. 3,847,822 (to Shuey); 3,802,842 (to Lange); 3,964,871 (to Hochstrasser); and 4,160,008 (to Fenochetti).
U.S. Pat. No. 3,802,842 (to Lange et al) describes a test strip incorporating a dry chemistry reagent system in which a sample receptive surface of an indicator (reagent) layer is covered by a fine mesh. The indicator layer can be supported upon a "colorless" and "transparent" support. The addition of the fine mesh to this test element reportedly results in enhancement in speed and uniformity of distribution of sample upon the surface of the indicator layer. This uniformity of distribution also reportedly results in substantial improvement in reproducibility of result.
U.S. Pat. No. 3,847,822 (to Shuey) describes what he terms an "asymmetric" membrane comprising a polymer blend of polyvinyl pyrolidone and cellulose acetate. The composition of the membrane reportedly has improved transport of blood solutes; notably insulin, while being substantially impermeable to albumin.
U.S. Pat. No. 3,964,871 (to Hochstrasser) describes a disposable indicator (test strip) having a built-in color intensity scale which is directly correlated to analyte concentration with a test sample. Accordingly, it is reportedly possible to simply immerse this indicator within a test sample and observe the progressive development in color within the various regions of the device. The development of color (indicator) within a specific region of the test strip can thus be directly correlated with a specific concentration of analyte.
U.S. Pat. No. 4,160,008 (to Fenochetti) describes a test device for performance of a clinical analysis of a sample for different analytes on a common support. The inventor has effectively isolated each distinct analytical test in separate zones on the common support by elevating the reagent specific layer above the common support and providing a blotter on the support to insure against run-off and cross-contamination of one analytical site by another.
The next generation (at least in terms of complexity) of dry chemistry reagent systems which has evolved is a multiple layered element, having at least three discrete functional layers. These discrete functional layers are a spreading layer, a reagent layer and a signal (indicator) layer.
The performance of glucose determinations on whole blood, utilizing multiple layered films, is the subject of numerous publications and issued patents. The following listing is representative of the technical publications in this area: Walter, B., Dry Reagent Chemistries in Clinical Analysis, Analytical Chemistry, Vol. 55, No. 4, pp. 498-514 (April, 1983); Curme, Henry G., et al., Multilayer Film Elements for Clincal Analysis: General Concepts, Clinical Chemistry, Vol. 24, No. 8, pp. 1335-1342 (August, 1978); Spayd, Richard W., et al., Multilayer Film Elements for Clinical Analysis: Applications to Representative Chemical Determinations, Clinical Chemistry, Vol. 24, No. 8, pp. 1343-1350 (August, 1978); Ohkubo, Akiyuki, et al., Plasma Glucose Concentrations of Whole Blood, as Determined with a Multilayer-Film Analytical Element, Clinical Chemistry, Vol. 27, No. 7, pp. 1287-1290 (July, 1981); Ohkubo, Akiyuki, et al., Multilayer-Film Analysis for Urea Nitrogen in Blood, Serum, or Plasma, Clinical Chemistry, Vol. 30, No. 7, pp. 1222-1225 (July, 1984); and, Rupchock, Patricia, et al., Dry-Reagent Strips Used for Determination of Theophylline in Serum, Clinical Chemistry, Vol. 31, No. 5, pp. 737-740 (May, 1985). The following listing is representative of the corresponding patent literature in this area: 4,042,335 (to Clement); 4,059,405 (to Sodickson, et al); 4,144,306 (to Figueras); 4,258,001 (to Pierce); and, 4,366,241 (to Tom, et al).
U.S. Pat. No. 4,042,335 (to Clement), describes a series of multilayered analytical element suitable for performing chemical analysis of whole blood samples. The Clement configurations all contemplate the application of test samples either directly, or from a spreading layer, onto a reagent layer. The reagent layer contains a complement of chemicals for reaction with a specific analyte suspected of being present in the test sample. If the analyte is present, a "detectable species" is formed or released from the reagent layer and diffuses into what is termed a "registration layer"--that is, a layer whose sole function is to provide a medium or repository from which the detectable species can be observed or measured. In order to avoid interference (masking) in the observation or measurement of the detectable species, the registration layer is both devoid of the test sample and reagents used in the generation of the diffusible species. In the preferred embodiments of the Clement test element, an optical screen ("radiation blocking layer") is also provided between the reagent layer and the registration layer. This optical screen effectively optically isolates the detectable species from other constituents which could interfere in its detection and/or measurement.
As is evident from the foregoing description, Clement attempts to segregate the individual functions of his analytical element into discrete layers. This technique, although potentially attractive to a manufacturer in possession of technology for fabrication of multilayered elements, is by its very nature unduly complex and potentially troublesome due to the mechanical instability of these composites. More specifically, where this test element is to be used by an individual in a self-test environment, the composite must necessarily be supported on an additional element to lend physical integrity to the multi-layered element and thereby prevent its unintended flexing and potential separation of the various layers contained therein.
U.S. Pat. No. 4,059,405 (to Sodickson, et al), describes a method and apparatus for glucose analysis of whole blood samples. In the Sodickson system (as described in Example 1 d.), a reaction site is initially prepared by preforming wells in a polyox resin treated filter paper. A reaction site is physically defined in this treated paper by impressing thereon a confining ring approximately one centimeter in diameter. A glucose reagent is then applied to the reaction site defined by this ring and the reaction site dried. An ultrafiltration membrane is placed over the well and a sheet of paper containing a dried blood spot placed in contiguous relationship with the ultrafiltration membrane. The dried blood spot is then reconstituted by the addition of saline. The apparatus used in the Sodickson system (i.e. press) confines the reconstituted blood sample in the reaction well for a brief incubation period. During this incubation period , soluble components of the whole blood sample are redissolved in the saline and pass through the ultrafiltration membrane where they come in contact with the glucose test reagents in the polyox treated paper. The cellular components of the blood are retained on the ultrafiltration membrane and thereby prevented from interference with the development and/or measurement of the glucose manifesting indicator.
The system described by Sodickson, as contemplated in his Example 1 d., is cumbersome (requiring reconstitution of the blood sample and relatively complex equipment to effect separation of cellular components from the whole blood sample) and does not readily lend itself to self-testing.
U.S. Pat. No. 4,144,306 (to Figueras), describes a multi-layered analytical element analogous to that of the Clement patent (previously discussed). The Figueras chemistry differs from Clement in that the interaction of an analyte and non-diffusible reagents in the reagent layer, results in the release of a "preformed detectable species" which can migrate from the reagent layer into a registration layer. This preformed detectable species is then observed or measured in the registration layer. Figueras contemplates (as described in his Example VI) the adaptation of his system to glucose analysis of whole blood. The separation of colored and cellular components of the whole blood would be achieved by Figueras in essentially the same fashion as in the Clement patent. The introduction of the whole blood sample into the reagent layer of the Figueras element results in the release of a diffusible preformed photographic dye, which is then free to migrate into the registration layer. Figueras requires the presence of the same type of optical screen (radiation blocking layer) between the reagent layer and the registration layer to avoid masking or interference in detection of the dye form the non-diffusible color components (i.e. sample and reagents) in the reagent layer. The limitations and disadvantages noted in the discussion of the Clement patent are also applicable to the multi-layered analytical element of Figueras. Figueras, however, introduces an additional complexity; namely, the effective immobilization of the reagents within the reagent layer and the preservation of the preformed indicator prior to its release by the analyte of interest. Because of the requirements of maintenance of fluid contact between the various elements of the Figueras composite, its mechanical properties are critical. Accordingly, the multi-layered element of Figueras, as previously noted for Clement, will require a supporting (transparent) layer to lend physical integrity to this device.
U.S. Pat. No. 4,258,001 (to Pierce et al), describes a multi-layered analytical element (of the type described in both Clement and Figueras--previously discussed) incorporating a unique spreading layer. The spreading layer of the Pierce patent is described as an essentially "non-fibrous" material. In one of the preferred embodiments described by Pierce (Figure III), the spreading layer can contain "interactive compositions" (test reagents) for reaction with analytes in a test sample. Pierce also contemplates the use of her device in the analysis of whole blood, blood serum and urine. Whole blood can be applied directly to the Pierce element. The presence of red blood cells will not reportedly interfere with spectrophotometric analysis if carried out by reflectance measurements, provided a radiation screen (blocking layer) is used to screen out interference from the red cells (column 26, line 49-61).
As is evident from the above patent, the Pierce device is designed to "take up" the whole blood sample. Thus, both cellular and non-cellular components of the whole blood are imbibed by the spreading layer. The spreading layer of Pierce is, therefore, not intended nor contemplated as a means for separation of the cellular fraction of the blood from the serum fraction. Where enzyme based diagnostic clinical assays are incorporated into the spreading layer (as in the case of glucose analysis), the potential for inhibition of these enzymes by the erythrocytes can potentially mask low concentrations of glucose and, thus, distort an otherwise clinically significant result.
The transport and spreading of biological samples is of concern, not only in dry chemistry reagent systems for performance of clinical assay, but also in the dry reagent systems utilized for immunoassay of the same biological fluids. The following U.S. Patents are representative of the immunoassay literature in this area: U.S. Pat. Nos. 4,094,647 (to Deutsch); and, 4,366,214 (to Tom et al).
U.S. Pat. No. 4,094,647 (to Deutsch), describes a linear wick having defined areas for placement of reagents and sample. The end of the wick is placed in a vertical position in a developer fluid and the fluid drawn up the wick by capillary action. As the fluid is drawn into the wick, it transports the reagents and sample, from their respective locations, into contact with one another. One or more of these reagents can be immobilized on the wick; thus, the developer fluid is used to transport reagent and sample to the immobilized reagent and any unreacted or mobile materials from the immobilized reagent. The site having the immobilized reagent can then be viewed or measured for the presence of analyte.
U.S. Pat. No. 4,366,241 (to Tom et al) describes a device for the nonchromatographic immunoassay of biological fluids. In the Tom device configuration, a test element, having a relatively small test zone, is treated with an immobilized binding material (termed "mip" or "member of an immunological binding pair"). The test zone of this device is the exclusive entry port for the biological sample and is designed for receipt of the biological sample either by direct application or immersion in the test fluid. The analyte (if any) contained in the biological sample is selectively (immunochemically) bound in the test zone to the immobilized binding material which is specific for this analyte. The residual components of the sample, including the fluid component thereof, are drawn from the test zone to a second element which is in fluid contact (contiguous relationship) with the test zone. The second element's function is to pump or draw the biological sample through the test zone into the test element. Those constituents of the sample which are not bound in the test zone are, thus, drawn into the test element and away from the test zone.
In the immunological test element of the type described by Tom, the pretreatment of the test zone effectively confines the analyte and the test reagents (i.e. labelled indicator) to the analysis site, thereby essentially eliminating the problems of reagent and sample migration (which are common in the solid phase systems designed for clinical chemistry analysis). These immunoassay systems of Tom are not, however, without their disadvantages, the most common being the nonspecific binding of interfering substances in the reaction zone and the difficulties which are sometimes inherent in the detection of low levels of analyte.
U.S. Pat. No. 4,446,232 (to Liotta) describes a multiple layer test device for immunoassay. This device and test protocol are directly analogous to the Figueras patent (previously discussed). The addition of sample containing an analyte of interest results in the displacement of a previously immobile species. In the system described by Liotta, the component of the reagent system which is displaced is an enzyme conjugated to a member of an immunological binding pair (hereinafter "conjugate"). The conjugate is then carried into the laminate where it can effect production of a detectable species.
As is evident from the foregoing discussion of the references appropriate for whole blood analysis, each type of test element generally requires a plurality of lamina in its preferred configuration. Where a single layer (component) test device is suggested, none of the references, with one exception (U.S. Pat. No. 3,607,093-to Stone), either acknowledge or appreciate the potential chemical and optical interference of the erythrocyte population (and other colored components of the blood), on the analytical protocol or in the detection of the reaction product which is indicative of the presence of the analyte (namely, the glucose). Where immobilization techniques (as described in Fetter, Deutsch and Tom) are employed, the specificity of the binding reaction can, under certain conditions, be indiscriminate. In the case of Fetter, the attempt at scavenging of erythrocytes and other colored components from the sample with certain salts (that have been imbibed within the test medium), is not without its limitations. In the single layered element of Fetter, the serum fraction is radially separated from the colored component of the blood and thereby results in the distribution of the analyte over a relatively large area. If the analyte is present in low concentrations, it can easily escape detection. Thus, some amplification mechanism may be required for visualization of a low level of analyte (i.e., the use of an enzyme in conjunction with a chromogenic substrate).
The immunochemical device and techniques of the type described by Deutsch, Tom amd Liotta are not readily compatible with whole blood analysis. It is possible to wash the test element for removal of the cellular debris (as is suggested in the Free patent), however, the effect of such additional step upon the immunochemical binding process is not known and can be expected to introduce analytical error in such analysis. Such manipulations of the test element can also be expected to mask detection of the analyte manifesting reaction or removal (in part) of the indicator species which is indicative of the analyte of interest. In addition, such physical removal of cellular and colored debris will necessarily expose the clinician or the person performing the test to potential infection by those components of the blood which are removed from the test element.
In summary, it should now be apparent that the prior art lacks a simple yet accurate device for analysis of whole blood. Where such devices have been proposed, they are complex, do not readily lend themselves to self-testing without the provision of a fixture or additional support mechanism, and generally lack the sensitivity to permit differentiation of different levels of analyte over a broad clinical range of concentration. Moreover, all such devices discussed above (with the exception of the Stone patent) would appear to have one common failing; namely, their inability to effect optical (and in certain instances, chemical) isolation of erythrocyte and other colored components of the whole blood from the observation/measurement site without the physical removal of the erythrocytes from the test element; or, the provision of some optical screen (blocking layer) between the colored components of the sample and the indicator compound which is generated as a result of the clinical assay.